The use of biomass as a renewable source of energy has become a major strategy for replacement of current energy matrix based on fossil fuels. The main constituent of biomass is cellulose that is basically comprised of glucose molecules covalently linked by b-1,4 linkages. However, it is extremely recalcitrant to degradation by enzymatic routes. For hydrolysis of the cellulosic fraction are required at least three enzymatic classes: endocelulases, exocelulases and ²-glucosidases. Recent studies have demonstrated novel proteins that assist in the degradation of cellulose such as swollenins (participate in the amorphogenesis of the lignocellulosic material) and redox enzymes (oxidize the C-H bond at the position C1 of the saccharide). Additionally, cellulases commonly exhibit a modular architecture strictly composed of a catalytic domain, which may be accompanied by one or more accessory domains, which assist in the recognition of substrate and increase the catalytic efficiency. The most common ancillary domains are carbohydrate-binding modules (CBMs), fibronectin-like and immunoglobulin-like (Ig-like) domains - the function of the latter is still not fully understood. The Ig-like is found in some enzymes belonging to the family GH9 that have a characteristic fold of the catalytic domain (±/±)6. Despite advances in the area and the many existing strategies for increasing the catalytic efficiency of cellulases, the cellulase activity still represents one of the major bottlenecks for the saccharification of lignocellulosic material in an economically viable manner. Thus, the objective of this project is to investigate two novel cellulases isolated from the plant pathogen Xanthomonas axonopodis pv . citri which has an arsenal of glycoside hydrolases in untapped biotechnological standpoint . In particular, we aim functional and structural studies of a cellulase belonging to the family GH5 that has no accessory domains and has low sequence identity with other characterized enzymes (identity ~ 35%) and a modular GH9 enzyme containing a Ig -like domain, which its function is still elusive. Preliminary studies are very promising indicating that the GH5 enzyme shows thermal stability exacerbated in the presence of the ion Mn2+, but the binding site described in cellulase 5A from Bacillus subtilis (Santos et al. 2012) is not conserved indicating another structural determinant. Besides seeking to identify the site responsible for such stability, this project intends to pursue other strategies to gain stability and increased catalytic efficiency as insertion of glycosylation sites and site-directed mutations drawn from structural data. Collectively, these studies will describe the biotechnological potential of cellulases from pathogen X. axonopodis pv. citri and expand knowledge on molecular mechanisms to gain stability in glycoside hydrolases that can have great impact on industrial processes that use biomass as feedstock.
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